Recently, non-thermal plasma (NTP) technologies have found widespread application across diverse fields, including plant growth, medical science, and biological and environmental research. Rice (Oryza sativa L.) is exceptionally sensitive to temperature changes. Notably, low-temperature stress primarily affects the germination and reproductive stages of rice, often leading to reduced crop yield. This study aimed to identify optimal conditions for enhancing rice seed germination and seedling growth under low temperatures using NTP technology. Our research indicated that NTP treatment at 15.0 kV for 30 s optimally promotes rice seed germination and growth under low-temperature stress. Furthermore, NTP treatment increases the activity and expression of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), under low-temperature conditions. Moreover, it downregulates the expression of β-ketoacyl-[acyl carrier protein] synthase I (KASI) and cis-epoxy carotenoid dioxygenase 3 (NCED3) and upregulates the expression of alternative oxidase (AOX1B), BREVIS RADIX-like homologous gene (BRXL2), WRKY transcription factor 29 (WRKY29), and EREBP transcription factor 2 (EREBP2) in roots after tandem 7 days low-temperature (16 ℃) and 7 days room-temperature (28 ℃) treatments. Transcriptomic analysis revealed the involvement of various key genes in phosphotransferase activity, phosphate-containing compound metabolic processes, and defense responses. These analyses provide comprehensive information on gene expression at the transcriptional level, offering new insights for a deeper understanding of candidate genes required for root growth in rice.